scholarly journals Highly Hydrolytic Reuteransucrase from Probiotic Lactobacillus reuteri Strain ATCC 55730

2005 ◽  
Vol 71 (7) ◽  
pp. 3942-3950 ◽  
Author(s):  
S. Kralj ◽  
E. Stripling ◽  
P. Sanders ◽  
G. H. van Geel-Schutten ◽  
L. Dijkhuizen
Keyword(s):  
Lactobacillus Reuteri ◽  
Activity Ratio ◽  
Probiotic Strain ◽  
Transferase Activity ◽  
Strain Atcc ◽  
Isolation And Characterization ◽  
Branching Points ◽  
Probiotic Lactobacillus ◽  
Culture Supernatants

ABSTRACT Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (∼70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.

scholarly journals Molecular Characterization of a Novel Glucosyltransferase from Lactobacillus reuteri Strain 121 Synthesizing a Unique, Highly Branched Glucan with α-(1→4) and α-(1→6) Glucosidic Bonds

2002 ◽  
Vol 68 (9) ◽  
pp. 4283-4291 ◽  
Author(s):  
S. Kralj ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
R. J. Leer ◽  
E. J. Faber ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Lactobacillus Reuteri ◽  
Degenerate Primers ◽  
Terminal Domain ◽  
Branching Points ◽  
Culture Supernatants

ABSTRACT Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the α-(1→4) glucosidic type. The glucan also contains α-(1→6)-linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M r of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

scholarly journals Isolation Detection and Characterization of Syringolin A produced from the Probiotic Strain Bacillus Cereus Isolated from Donkey Milk

2018 ◽  
Vol 1 (2) ◽  
Author(s):  
Rashmi D ◽  
Sharmila T ◽  
Sushama Patil ◽  
Onkar Apine ◽  
Srinivas Sistla ◽  
...  
Keyword(s):  
Probiotic Strain ◽  
Uv Spectra ◽  
Organic Layer ◽  
Characteristic Peak ◽  
Antitumor Compound ◽  
Solvent Systems ◽  
Culture Supernatants ◽  
Layer Chromatography ◽  
Economical Process

Syringolin A is a non-ribosomal virulence factor secreted by few Pseudomonas strains. Syringolin A is an well known irreversible proteasome inhibitor and antitumor compound. The present study is focused on the extraction of Syringolin A through a non-tedious and economical process. Syringolin A is extracted from culture supernatants by the immiscible organic layer by mixing of dichloromethane or chloroform (trichloromethane). Syringolin A was identified by the characteristic peak at 350 nm by UV spectra. The compound was further characterized by Thin Layer Chromatography (TLC) with the retention value, Rf was found to be in the range of 0.78-0.83 run using a combination of solvent systems water and methanol.  The molecular weight of the compound was found to be 492.2614 g mol-1 identified and analyzed by UHPLC–QTOF-MS analysis. Due to its significant pharmacological importance in proliferative diseases, further studies on production and optimization of these compounds are necessary.   

scholarly journals Isolation and Characterization of an Autoinducer Synthase from Acinetobacter baumannii

2008 ◽  
Vol 190 (9) ◽  
pp. 3386-3392 ◽  
Author(s):  
Chen Niu ◽  
Katy M. Clemmer ◽  
Robert A. Bonomo ◽  
Philip N. Rather
Keyword(s):  
Acinetobacter Baumannii ◽  
Homoserine Lactone ◽  
Transcriptional Level ◽  
Human Pathogen ◽  
Isolation And Characterization ◽  
Opportunistic Human Pathogen ◽  
Biofilm Development ◽  
Culture Supernatants ◽  
Ethyl Acetate Extracts

ABSTRACT The opportunistic human pathogen Acinetobacter baumannii strain M2 was found to produce distinct acyl-homoserine lactone (AHL) signals based on the use of an Agrobacterium tumefaciens traG-lacZ biosensor. An A. baumannii gene, designated abaI, was cloned and directed AHL production in recombinant Escherichia coli. The AbaI protein was similar to members of the LuxI family of autoinducer synthases and was predicted to be the only autoinducer synthase encoded by A. baumannii. The primary AHL signal directed by AbaI was identified by mass spectrometry as being N-(3-hydroxydodecanoyl)-l-HSL (3-hydroxy-C12-HSL). Minor amounts of at least five additional AHLs were also identified. The expression of abaI at the transcriptional level was activated by ethyl acetate extracts of culture supernatants or by synthetic 3-hydroxy-C12-HSL. An abaI::Km mutant failed to produce any detectable AHL signals and was impaired in biofilm development.

scholarly journals Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2099-2112 ◽  
Author(s):  
S. Kralj ◽  
G. H. van Geel-Schutten ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Enzyme Kinetics ◽  
Lactobacillus Reuteri ◽  
Variable Domain ◽  
Transferase Activity ◽  
Catalytic Core ◽  
Total Enzyme Activity ◽  
Specific Activities

Lactobacillus reuteri strain 121 uses sucrose for synthesis of a unique, soluble glucan (‘reuteran’) with mainly α-(1→4) glucosidic linkages. The gene (gtfA) encoding this glucansucrase enzyme had previously been characterized. Here, a detailed biochemical and molecular analysis of the GTFA enzyme is presented. This is believed to be the first report describing reuteransucrase enzyme kinetics and the oligosaccharides synthesized with various acceptors. Alignments of the GTFA sequence with glucansucrases from Streptococcus and Leuconostoc identified conserved amino-acid residues in the catalytic core critical for enzyme activity. Mutants Asp1024Asn, Glu1061Gln and Asp1133Asn displayed 300- to 1000-fold-reduced specific activities. To investigate the role of the relatively large N-terminal variable domain (702 amino acids) and the relatively short C-terminal putative glucan-binding domain (267 amino acids, with 11 YG repeats), various truncated derivatives of GTFA (1781 amino acids) were constructed and characterized. Deletion of the complete N-terminal variable domain of GTFA (GTFA-ΔN) had little effect on reuteran characteristics (size, distribution of glycosidic linkages), but the initial transferase activity of the mutant enzyme increased drastically. Sequential C-terminal deletions (up to six YG repeats) in GTFA-ΔN also had little effect on reuteran characteristics. However, enzyme kinetics drastically changed. Deletion of 7, 8 or 11 YG repeats resulted in dramatic loss of total enzyme activity (43-, 63- and 1000-fold-reduced specific activities, respectively). Characterization of sequential C-terminal deletion mutants of GTFA-ΔN revealed that the C-terminal domain of reuteransucrase has an important role in glucan binding.

scholarly journals Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (1) ◽  
pp. 177-184 ◽  
Author(s):  
M E Dihanich ◽  
D Najarian ◽  
R Clark ◽  
E C Gillman ◽  
N C Martin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Transferase Activity ◽  
Isopentenyl Transferase ◽  
Nuclear Mutation ◽  
Isolation And Characterization ◽  
Isopentenyl Adenosine ◽  
Cellular Compartments

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

scholarly journals Proteomic characterization of a selenium-metabolizing probiotic Lactobacillus reuteri Lb2 BM for nutraceutical applications

PROTEOMICS ◽  
2018 ◽  
Vol 18 (13) ◽  
pp. 1870116
Author(s):  
Cristina Lamberti ◽  
Erika Mangiapane ◽  
Alessandro Pessione ◽  
Roberto Mazzoli ◽  
Carlo Giunta ◽  
...  
Keyword(s):  
Lactobacillus Reuteri ◽  
Probiotic Lactobacillus

Proteomic characterization of a selenium-metabolizing probiotic Lactobacillus reuteri Lb2 BM for nutraceutical applications

PROTEOMICS ◽  
2011 ◽  
Vol 11 (11) ◽  
pp. 2212-2221 ◽  
Author(s):  
Cristina Lamberti ◽  
Erika Mangiapane ◽  
Alessandro Pessione ◽  
Roberto Mazzoli ◽  
Carlo Giunta ◽  
...  
Keyword(s):  
Lactobacillus Reuteri ◽  
Probiotic Lactobacillus

scholarly journals Composition and Clinical Significance of Exosomes in Tuberculosis: A Systematic Literature Review

2021 ◽  
Vol 10 (1) ◽  
pp. 145
Author(s):  
Fantahun Biadglegne ◽  
Brigitte König ◽  
Arne C. Rodloff ◽  
Anca Dorhoi ◽  
Ulrich Sack
Keyword(s):  
Vaccine Development ◽  
Biological Fluids ◽  
Diagnostic Tools ◽  
Major Health ◽  
Fast Detection ◽  
Isolation And Characterization ◽  
Culture Supernatants

Tuberculosis (TB) remains a major health issue worldwide. In order to contain TB infections, improved vaccines as well as accurate and reliable diagnostic tools are desirable. Exosomes are employed for the diagnosis of various diseases. At present, research on exosomes in TB is still at the preliminary stage. Recent studies have described isolation and characterization of Mycobacterium tuberculosis (Mtb) derived exosomes in vivo and in vitro. Mtb-derived exosomes (Mtbexo) may be critical for TB pathogenesis by delivering mycobacterial-derived components to the recipient cells. Proteomic and transcriptomic analysis of Mtbexo have revealed a variety of proteins and miRNA, which are utilized by the TB bacteria for pathogenesis. Exosomes have been isolated in body fluids, are amenable for fast detection, and could contribute as diagnostic or prognostic biomarker to disease control. Extraction of exosomes from biological fluids is essential for the exosome research and requires careful standardization for TB. In this review, we summarized the different studies on Mtbexo molecules, including protein and miRNA and the methods used to detect exosomes in biological fluids and cell culture supernatants. Thus, the detection of Mtbexo molecules in biological fluids may have a potential to expedite the diagnosis of TB infection. Moreover, the analysis of Mtbexo may generate new aspects in vaccine development.

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